Chapter 5: Biological and Solubility Evaluation of the Substituted Ajoenes
5.4 Ajoene analogues as Chemosensitizing agents
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69 delivery into the tumours when it is administered via intraperitoneal injection. However, further investigation needs to be carried out to support this hypothesis and to also evaluate exactly which receptors are likely responsible for the interaction observed. A study conducted by Apitz-Castro showed that ajoene (with the same vinyl disulfide/sulfoxide pharmacophore) has affinity towards the fibrinogen receptor, which is found in human platelets and prevents platelet aggregation. Thus, it is likely that the bis-PMB might bind to similar receptors reversibly, but this is still to be proven.55
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70 Table 4: Cytotoxicity against hematologic and solid tumor cell lines°.
Compound CRL-
7065
a CC50
DU-145
bCC50
Hela
cCC50
HEP-
dCCG2 50
SK-MEL-
eCC28 50
SK-MES-1
fCC50
gCEM CC50
hCCSB 50
KBWT
iCC50
KBMDR
jCC50
KB7D
kCC50
KBV20C
LCC50
(E) Ajoene, 4 >30 >30 >30 >30 >30 >30 5.4 ± 0.5 6.3 30.8 ± 5 50.7 ± 17.2 35.8 ± 1.6
(Z) Ajoene, 4 >30 >30 >30 >30 >30 >30 6.2 ± 0.7 6.4 22.0 34.1 ± 12.4 22.0 ± 2.4
(E)Propyl-Allyl, 30 >30 >30 >30 >30 >30 >30 2.3 ± 0.2 5.7 13.2 ± 1.1 28.8 ± 5.5 18.8 ± 1.1
(Z)Propyl-Allyl, 30 >30 >30 >30 >30 >30 >30 3.1 ± 0.4 5.7 23.1 ± 2.6 34.9 ± 5.3 23.3 ± 1.3
(E) Bis-Propyl, 31 >30 29.45 >30 >30 >30 >30 2.6 ± 0.5 6.0 19.6 37.7 ± 10.9 23.6 ± 1.2
(Z) Bis-Proply, 31 >30 29.32 >30 >30 >30 >30 2.6 ± 0.5 3.2 14.4 ± 1.7 31.3 ± 3 16.7 ± 2.3
(E/Z) PMB-Allyl, 32 >30 16.4 27.46 >30 >30 >30 1.3 ± 0.4 3.5 7.8 ± 0.6 13.8 ± 1.9 8.5 ± 1.7
(E/Z) Bis-PMB, 5 >30 11.83 13.34 >30 >30 >30 0.8 ± 0.2 0.2 4.5 ± 1.3 7.4 ± 1.5 5.4 ± 1.0
(E/Z) PMB-Acetate,
27 >30 >30 9.65 >30 >30 >30 0.8 ± 0.1 3.1 3.5 ± 1.3 8.2 ± 0.7 4.8 ± 1.3
(E/Z) PMB-Ester, 28 >30 >30 >30 >30 >30 >30 5.4 5.1 11.9 ± 1.7 21.8 ± 2.1 12.1 ± 1.3
(E/Z) PMB-Phenol,
26 >30 >30 >30 >30 >30 >30 0.8 ± 0.1 3.1 4.4 ± 1.3 8.0 ± 1.2 5.4 ± 1.2
p6-MP 2.1±0.4 1.4±0.7
qCDDP 1.0±0.2 1.4±0.2
Vincristine 0.015 2.6 >4 0.015 0.004±0.0008 0.012±0.0003 0.004±0.00
2 0.95±0.2 0.09±0.01 0.40±0.08
Doxorubicin 0.44 0.3 1.1 0.21 0.12±0.06 0.05±0.03 0.4±0.08 ≥20 3.3±1 2.2±0.7
rEtoposide 4.6 7.6 7.8 2.04 3.3±1.1 ≥100 ≥100 7.6±2.5
°Data represent mean values for three independent determinations.
aCC50 : Compound concentration (µM) required to reduce the viability by 50%, as determined by the MTT method.
bCRL-7065: skin fibroblasts
cDU-145: human prostate carcinoma;
dHela: human cervical adenocarcinoma;
eHEP-G2: human hepatocellular carcinoma;
fSK-MEL-28: human skin malignant melanoma;
gSK-MES-1: human squamous cell lung carcinoma;
hCCRF-CEM: human acute T-lymphoblastic leukemia;
iCCRF-SB: human acute B-lymphoblastic leukemia;
lKBWT: human nasopharyngeal carcinoma, wild type;
mKBMDR: human nasopharyngeal carcinoma, multidrug resistent infected with a retroviral vector containing a full-length cDNA for the human MDR1 gene that conferred the full MDR phenotype;
nKB7D: human nasopharyngeal carcinoma, stepwise selection for resistance with increasing concentration of etoposide;
oKBV20C: human nasopharyngeal carcinoma, stepwise selection for resistance with increasing concentration of vincristine;
p6-MP: 6-mercaptopurine
qCDDP: Cis-platinum
rEtoposide: VP16
[ ] = increased cytotoxicity [fold]
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71 The results showed both a synergistic and also an antagonistic effect when the analogues were mixed with the drugs DOXO and VCR. When compounds (E/Z)-ajoene 4, 30 (propyl - allyl) and the (E/Z)-bis-propyl 31 analogues were mixed with the two drugs and tested against the KBV20C cell-line which has resistance to VCR the results indicated an increase in resistance, i.e.
negative chemosensitization. Reasons for this antagonism are unclear. The opposite effect was observed for the PMB-allyl 32 to the PMB-Phenol 26. For the former, the CC50 of Vincristine (the concentration required to reduce viability by 50% of the KBV20C cells) was 0.85 M which decreased by eighteen fold to 0.047 M when 6.67 M of the PMB-allyl was combined with VCR. Of importance is that 6.67 M was below the CC50 of the PMB-allyl 32 of 8.5 M, meaning that the reduced CC50 of the drug in combination was not due to the ajoene derivative. This result indicated a synergy (positive chemosensitization) as it had been shown that the analogue alone had a CC50 of 8.5 M (see Table 4 and Figure 32).
Figure 32: Representation of the chemosensitization effect of Vincristine and PMB-allyl against a VCR-resistant cancer cell-line.
In the case when the PMB-allyl concentration was lowered to 2.22 M the required VCR was 0.1 M showing that the synergistic effect as expected is ajoene-concentration dependent. This was still a healthy reduction in the CC50 of the drug.
Similarly, the PMB-Ester 28 showed positive chemosensitization towards both DOXO (Doxorubicin) as well as VCR in KBWT (human nasopharyngeal carcinoma, wild type), with significant reductions in CC50 of the drugs at 8.33 M of 28 which is below the CC50 of 28 (11.9
M) in this cell-line, see Figure 33.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
alone 20µM 6,67µM 2,22µM
VCR w/ MM4
CC50 [M]
KB V20C: CC50 [ ] VCR IN THE ABSENCE AND IN THE PRESENCE OF PMB-Allyl, 32
TOXIC
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72 Figure 33: The comparison of CC50 when PMB-Ester 28 is combined with both DOXO and VCR in KBWT.
In addition, The PMB-phenol 26 showed significant chemosensitization activity at 3.33 M against the VCR-resistant cell-line KBV20C, and importantly at a concentration below its own CC50 (5.4 M in this cell-line, see Table 4 and Figure 34). In this regard it is useful to have a derivative with a relatively high CC50.
0 0.1 0.2 0.3 0.4 0.5 0.6
alone 25µM 8,33µM 2,78µM
DOXO w/ MM7
CC50 [µM]
KB wt: CC50 [µM] DOXO IN THE ABSENCE AND IN THE PRESENCE OF PMB-Ester, 28
TOXIC
0 0.001 0.002 0.003 0.004 0.005 0.006 0.007
alone 25µM 8,33µM 2,78µM
VCR w/ MM7
CC50 [µM]
KB WT: CC50 [µM] VCR IN THE ABSENCE AND IN THE PRESENCE OF PMB-Ester, 28
TOXIC
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73 Figure 34: The comparison of CC50 when PMB-Phenol is combined with both DOXO and VCR.
These results show how addition of ajoene analogues switch on the sensitivity of the tumour cells towards the drug resulting in a reduction in the concentration of VCR required to achieve 50% inhibition. Mechanistic and structural reasons for this are unclear at this stage but an empirical observation is that all positive ajoene chemosensitizers had at least one p- methoxybenzyl group in their structure. This observation aids in the future design of chemosensitizing agents.12b, 30
0 0.5 1 1.5 2 2.5 3 3.5
alone 10µM 3,33µM 1,11µM
DOXO w/ MM8
CC50 [µM]
KB v20c: CC50 [µM] DOXO IN THE ABSENCE AND IN THE PRESENCE OF PMB-Phenol
TOXIC
0 0.02 0.04 0.06 0.08 0.1
alone 10µM 3,33µM 1,11µM
VCR w/ MM8
CC50 [µM]
KB V20C: CC50 [µM] VCR IN THE ABSENCE AND IN THE PRESENCE OF PMB-Phenol
TOXIC
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74